Design And Fabrication Of Label-Free Analysis Methods And Microfluidic Paper-Based Analytical Devices

Recently,label-free analytical method has attracted lots of interests.Label-free analytical methods exhibited various advantages,such as no requirement of complicated labeling process,direct analysis ability,simple and short operation processes,fast,and low-cost,thus has become a new trend in the field of analysis.Microfluidic paper-based analytical devices??PAD?which combined the advantages of test papers and the capability of lab-on-chip devices are first developed in recent years.Integrating of sample collection,pretreatment,directional transport,specific reaction,and detection together on one minimized device,?PAD has the advantages of low-cost,potable,simple,reagent saving,fast,biocompatible,and can be used for multiple analysis.As a micro-total analysis system,?PAD can be used as sensing platform for label-free analysis.This dissertation focuses on the development of novel label-free analytical methods and the construction of?PAD for multiplexed label-free analysis.A novel,simple,rapid,sensitive and cost effective label-free colorimetric method for the selective determination of GSH over Cys and Hcy was proposed.In this assay,firstly,o-phenylenediamine?OPD?could be oxidized by silver ions?Ag⁺?to produce silver nanoparticles?AgNPs?and pale yellow colored 2,3-diaminophenazine?shorted as OPDox?with absorbance peak center at 429 nm.The as-formed AgNPs could further catalyze the redox reaction between OPD and Ag⁺.In the presence of GSH,GSH could chelate with Ag⁺,inhibiting the oxidation ability of Ag⁺,and link with AgNPs,influence the catalytic ability of AgNPs.Furthermore,GSH possesses strong reducibility to reduce OPDox,resulting in color fading of the detection solution and decrease in absorbance intensity.The proposed Ag⁺-OPD based sensing system exhibited a wide linear range from 2 nM to 1?M for GSH detection,with the limit of detection as low as 1.7 nM.Moreover,this developed method was successfully applied to GSH detection inplasma and urine samples with satisfied results.A double-layered three-dimensional?3D??PAD with high resolution time-resolved chemiluminescence?CL?emissions were designed.Using a filter paper as a hydrophilic substrate,a novel compact 3D?PAD as prepared by virtue of wax-screen-printing technology.The fabricated 3D?PAD was composed of one injection zone and multiple detection zones.Using the luminol-H₂O₂ CL system as tested reaction system,four well-separated CL peaks were obtained,which verified that the designed 3D?PAD has high resolution time-resolved CL detection performance.A label-free CL detection sensor was developed for multiplexed determination of glucose,lactic acid,cholesterol and choline based on the fabricated?PAD.The sample was added to four detection zone modified with CL catalyst cobalt ion and different oxidase by virtue of chitosan.When luminol flowed to?PAD,four time-resolved CL peaks were successively generated from the cobalt ion catalyzed CL reactions between luminol and generated H₂O₂ from the specific enzymatic reactions between the oxidase and the analytes.The generated four CL emission peaks in the CL kinetic curve increased in proportion to the concentrations of glucose,lactate,cholesterol,and choline,respectively.Finally,four linear calibration curves were obtained for the detection of glucose?0.01-1.0 mmol/L?,lactate?0.02-5.0 mmol/L?,cholesterol?0.01-0.4 mmol/L?,and choline?0.001-1.0 mmol/L?.The detection limits were as low as 8?mol/L,15?mol/L,6?mol/L,and 0.07?mol/L for glucose,lactate,choline,and cholesterol detection,respectively.The present work provides a new strategy for the fabrication of simple and sensitive 3D?PAD with high resolution time-resolved CL emissions for multiplexed chemiluminescence analysis,which holds great application potential for clinical diagnosis.